Research progress in toxicological effects and mechanism of aflatoxin B1 toxin

Fungal contamination of animal feed can severely affect the health of farm animals, and result in considerable economic losses. Certain filamentous fungi or molds produce toxic secondary metabolites known as mycotoxins, of which aflatoxins (AFTs) are considered the most critical dietary risk factor for both humans and animals. AFTs are ubiquitous in the environment, soil, and food crops, and aflatoxin B1(AFB1) has been identified by the World Health Organization (WHO) as one of the most potent natural group 1A carcinogen. We reviewed the literature on the toxic effects of AFB1 in humans and animals along with its toxicokinetic properties. The damage induced by AFB1 in cells and tissues is mainly achieved through cell cycle arrest and inhibition of cell proliferation, and the induction of apoptosis, oxidative stress, endoplasmic reticulum (ER) stress and autophagy. In addition, numerous coding genes and non-coding RNAs have been identified that regulate AFB1 toxicity. This review is a summary of the current research on the complexity of AFB1 toxicity, and provides insights into the molecular mechanisms as well as the phenotypic characteristics.


INTRODUCTION
Due to global climate change, mycotoxin-producing fungal strains that were endemic in the tropical-subtropical climate zones have also appeared in the temperate zones. Mycotoxins are secondary metabolites produced by filamentous fungi or mold present in the soil, grain, forage, and silage. They are non-essential for fungal growth and reproduction but are capable of inducing biochemical, physiological and pathological changes in many species (Wang et al., 2018a). The Food and Agricultural Organization (FAO) estimates that around 25% of the global agricultural productions and derived food products are contaminated with mycotoxins, which render an estimated 50 million tons of food inedible, resulting in severe economic losses each year (Iheshiulor et al., 2011). However, the current detectable rate of mycotoxins is as high as 60-80%, which is considerably higher than the FAO estimate of 25% (Eskola et al., 2020). Aflatoxin (AFT) is mainly produced by Aspergillus flavus and A. parasiticus, and is a derivative of difuranoxano-naphtho-ketone consisting of review, we have discussed the mechanisms under the toxic effects of AFB 1 in order to provide a reliable reference for further research in animal husbandry, mycotoxins, and the treatment of toxin-related diseases in humans, livestock and poultry.

TOXICOKINETICS OF AFB 1
AFB 1 exposure occurs through dietary intake, skin contact and inhalation of contaminated dust. More than 80% of the ingested AFB 1 is absorbed in the duodenum and the jejunum through passive transport (Grenier & Applegate, 2013), and accumulates thereafter in the liver, kidney and spleen, although the main target organ of AFB 1 is undoubtedly the liver. Toxic effects of AFB 1 have been observed in the liver, gastrointestinal tract, nervous system, immune cells, and reproductive organs.
Full-size DOI: 10.7717/peerj.13850/ fig-1 AFB 1 -N 7 -guanine have been detected in humans following AFB 1 exposure (Groopman et al., 1985;Ross et al., 1992;Mykkanen et al., 2005). The different AFB 1 metabolites are mainly expelled via feces and urine (Dohnal, Wu & Kuca, 2014). AFBO binds covalently to N 7 on guanine to form AFB 1 -N 7 -guanine adducts in the DNA double helix (Iyer et al., 1994), resulting in point mutations that may drive carcinogenesis (Lin et al., 2014). The common point mutation caused by the AFB 1 -N 7 -guanine adduct is a G →T transversion (Schermerhorn & Delaney, 2014;. Since exo-AFBO has a significantly higher affinity for guanine residues compared to endo-AFBO, it is considered to be the major carcinogenic metabolite. The AFB 1 -N 7 -guanine adduct forms an open ring structure under mild alkaline conditions, resulting in a stable AFB 1 -formamidopyridine adduct (AFB 1 -FAPy) (Smela et al., 2001) that is excreted with urine. Both isomers of AFBO are detoxified through glutathione (GSH) conjugation by glutathione-S-transferase. In addition, AFBO can be converted to AFT-mercapturic acid by GST, or to AFT-glucosiduronic acid by AFT-dihydropyridine, followed by the formation of GSH conjugates (Bryden, 2012). However, AFBO can also form adducts with serum albumin by covalently binding to the ε-amino group of lysine, which remains in circulation. Given its highly unstable nature, AFBO can spontaneously hydrolyze into AFB 1 dihydrodiol, which can cause tissue damage, inflammation, and excessive cell proliferation by conjugating with different proteins, eventually promoting carcinogenesis. One of the most common AFB 1 -induced mutations in human hepatocytes is a G → T transversion in codon 249 of the p53 gene, which causes a 249Arg → 249Ser mutation in the encoded protein (Foster, Eisenstadt & Miller, 1983;Hollstein et al., 1991;Soini et al., 1996). In addition, AFB 1 and AFBO can epigenetically increase the mutation rate of the p53 gene by methylating the CpG site in codon 248 (Narkwa, Blackbourn & Mutocheluh, 2017). The p53 gene is a tumor suppressor that is frequently mutated in human cancers, and the mutations promote tumor development by inhibiting apoptosis and increasing proliferation rates. AFBO can also induce mutations indirectly by binding to and damaging DNA repair enzymes (Weng et al., 2017). Furthermore, AFB 1 metabolism by the P450 enzyme also generates reactive oxygen species (ROS), such as hydroxyl free radicals, hydrogen peroxide and other free radicals that damage cell membrane and macromolecules (Kucukcakan & Hayrulai-Musliu, 2015).
The complex metabolic process of AFB 1 is a major determinant of its potent toxicity. Since the formation of DNA adducts by AFB 1 , and its metabolites can activate proto-oncogenes, quantitative analysis of AFB 1 -DNA adducts is an important indicator of AFB 1 toxicity.

EFFECT OF AFB 1 ON LIVESTOCK AND POULTRY
The susceptibility of livestock to AFB 1 differs across species. Monogastric animals are more susceptible to AFB 1 compared to the ruminants since the gut microbes in the latter can metabolize mycotoxins. Long-term exposure to low levels of AFB 1 is more common in livestock as opposed to acute poisoning (Fig. 2). Pigs are especially susceptible AFB 1 poisoning, and long-term exposure to low levels of dietary AFB 1 can inhibit their growth, impair digestive function, and disrupt the intestinal barrier by decreasing SOD activity, and increasing production of pro-inflammatory cytokines such as TNFα, IL1 and TGFβ (Pu et al., 2021). Nevertheless, there are reports of AFB 1 poisoning in ruminants as well.
Cattle fed with AFB 1 -spiked fodder showed behavioral changes such as depression and anorexia. AFB 1 significantly increased the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum creatinine (SCR), catalase (CAT) and malondialdehyde (MDA) in the affected cattle, and decreased that of total protein (TP), magnesium (Mg) and glutathione (GSH). Furthermore, autopsy of the poisoned cattle showed hepatomegaly, gallbladder enlargement, and intestinal and renal hyperemia (Elgioushy et al., 2020). AFB 1 contamination of poultry feed can reduce reproductive capacity, hatching rate, chick weight, growth rate, production rate and quality of meat and eggs, and increase susceptibility to diseases and mortality rate (Pandey & Chauhan, 2007;Fouad et al., 2019). In addition, the younger broilers were more susceptible to AFB 1 compared to older animals, which could be due to deficient detoxification mechanisms in the former (Wang et al., 2018b). In conclusion, exposure to AFB 1 can significantly affect the quality and productivity of livestock and poultry by altering their physiological and biochemical indices. Chronic exposure in particular leads to the accumulation of AFB 1 , and consumption of these contaminated products can adversely affect human health.

Hepatotoxicity
Liver is the main target organ for AFB 1 . Dietary supplementation of AFB 1 in rats lead to irreversible liver damage in a dose-dependent manner (Qian et al., 2016;Lu et al., 2013) by inducing fat deposition, fatty acid oxidation (Zhang et al., 2011) and telomere shortening (Ali et al., 2021). Recent studies have also shown that AFB 1 can trigger massive production of reactive oxygen species (ROS) in the liver cells, leading to oxidative stress, inflammation, and liver damage (Singh, Maurya & Trigun, 2015). Furthermore, AFB 1 exposure enhanced apoptosis of liver cells, activated the resident Kupffer cells, and promoted an inflammatory response in the liver through dephosphorylated-cyclooxygenase-2 (COX2) (Zhang et al., 2019a). Prolonged exposure to AFB 1 disrupted lipid and lipoprotein metabolism (Rotimi et al., 2017), and resulted in extensive damage to mitochondrial lipids and reduced antioxidant capacity in the rat liver (Rotimi et al., 2019). Likewise, mice exposed to AFB 1 showed     mitochondrial dysfunction and increased rates of mitochondria-dependent apoptosis in the liver (Xu et al., 2021). In one study, primary broiler hepatocytes (PBHs) treated with different concentrations of AFB 1 showed mitochondrial dysfunction, oxidative stress, and ROS-dependent mitochondrial apoptosis through the nuclear factor-erythroid 2-related factor-2 (Nrf2) signaling pathway (Liu & Wang, 2016). Epidemiological studies have shown that AFB 1 is one of the important risk factors of primary liver cancer (Kucukcakan & Hayrulai-Musliu, 2015). Furthermore, there is evidence that AFB 1 and chronic hepatitis B virus can synergistically induce mutations in the p53 gene and initiate liver cancer (Liu et al., 2012). To summarize, AFB 1 can induce liver damage and even liver cancer by inducing oxidative stress, inflammation, and mitochondrial dysfunction by targeting the p53, ROS, COX2, Nrf2 and other signaling pathways.

Enterotoxicity
The gut barrier function maintains the homeostasis between the resident immune cells and commensal microorganisms via the intestinal epithelial cells (IECs) (Peterson & Artis, 2014). Long-term exposure to AFB 1 has been associated with chronic intestinal diseases. AFB 1 caused intestinal mucosal injury and inhibited IECs proliferation in mice (Gaikwad & Pillai, 2004). In addition, AFB 1 and AFM 1 can damage the intestinal barrier in mice through clathrin-mediated endocytosis through synergistic and additive interactions (Gao et al., 2021). AFB 1 also altered the composition of the intestinal microbiota of male F344 rats in a dose-dependent manner, and significantly decreased the abundance of the probiotic lactic acid bacteria (Wang et al., 2016a). The microbial-related metabolic changes in the gut microbiota of these AFB 1 -treated rats were analyzed by ion fragmentation spectroscopy. AFB 1 significantly increased the number of inflammatory fecal liposomes, and altered intestinal microbiota-dependent biliary cholesterol metabolism, degradation of bilirubin and fatty acids, and glycolysis. The structural changes in the fecal microflora induced by AFB 1 are similar to that observed in IBD (inflammatory bowel disease) patients. The combination of metabolic dysfunction, loss of IECs and glandular atrophy caused by AFB1 can lead to chronic intestinal diseases (Zhou, Tang & Wang, 2021).
The gastrointestinal system in poultry is especially sensitive to AFB 1 . Broilers fed with different doses of AFB 1 exhibit severe damage to the intestinal villi characterized by lower density and absorption area (Yunus et al., 2011;Kana, Teguia & Tchoumboue, 2010), increased atrophy and shedding, and a significant reduction in height (Yin et al., 2016). Moreover, the jejunum of chickens exposed to AFB 1 showed histopathological changes, increased apoptosis rates, and altered expression levels of death receptors, endoplasmic reticulum (ER) molecules and apoptotic factors . Ingestion of AFB 1contaminated feed can also affect the absorption capacity of the small intestine and impair its innate immune function (Wang et al., 2018c). Furthermore, infiltration of inflammatory cells into the small intestine leads to muco-enteritis (Kumar & Balachandran, 2009). Ducks fed with AFB 1 -contaminated corns showed longer and wider jejunum villi, which was accompanied by lower average daily gain (ADG) and average daily feed intake (ADFI), resulting in reduced growth and development. Furthermore, the relative weight of the digestive organs, the activity of digestive enzymes and the biochemical indices of intestinal development were also altered (Feng et al., 2017). Thus, AFB 1 induced intestinal damage can restrict development, disturb the intestinal microflora, and lead to metabolic disorders or chronic intestinal diseases.

Nephrotoxicity
AFB 1 is absorbed by the kidneys, and its accumulation in the renal tissues leads to the upregulation of p21 by MYC, PLK1 and PLD1, resulting in S-phase cell cycle arrest and renal injury (Huang et al., 2019). HEK293 cells treated with AFB 1 showed increased apoptosis and DNA fragmentation, which corresponded to the up-regulation of p53, Bax and caspases (Dlamini et al., 2021). AFB 1 and AFM 1 synergistically increased oxidative stress and the apoptosis pathway in renal cells by regulating the expression level of L-proline (Li et al., 2018). Furthermore, the combination of AFB 1 exposure and low protein diet additively reduced weight gain and promoted renal dysfunction in rats, and exacerbated oxidative stress (Rotimi et al., 2018). Exposure to AFB 1 and DON synergistically increased oxidative stress in the liver, kidney, and spleen of carp by upregulating Nrf2. Interestingly, ROS generation occurred earlier in the kidneys compared to the liver and spleen (Kövesi et al., 2020). In conclusion, the nephrotoxicity of AFB 1 is mainly manifested as oxidative stress induced by p21, L-proline, Nrf2 and other genes. Moreover, AFB 1 can have synergistic nephrotoxic effects along with nutrient level and other mycotoxins.

Neurotoxicity
Intragastric administration of AFB 1 once weekly for 8 weeks significantly impaired brain function in rats by inducing pathological changes in the cerebral cortex and hippocampus (Bahey, Abd Elaziz & Gadalla, 2015). Long-term exposure to AFB 1 may allow it to penetrate the blood-brain barrier, resulting in neurotoxic effects and even chronic neurodegeneration such as that observed in Alzheimer's disease (Alsayyah et al., 2019). AFB 1 inhibits proliferation of human astrocytes by inducing cell cycle arrest and mitochondria-dependent apoptosis (Park et al., 2020). Environmental AFB 1 exposure may trigger neuroinflammatory responses by activating the microglia, and increase the susceptibility to neurodegenerative diseases (Mehrzad, Hosseinkhani & Malvandi, 2018a). The neurotoxic effects of AFB 1 exposure have also been observed in zebrafish embryos by nuclear magnetic resonance (NMR) (Zuberi et al., 2019). AFB 1 exposure decreased the survival rate of embryos by inhibiting oligodendrocyte development (Park et al., 2020). In addition, neuroblastoma cells (IMR-32 cell line) treated with AFB 1 also presented intracellular ROS accumulation, DNA damage, S phase arrest and apoptosis (Huang et al., 2020). To summarize, AFB 1 can inhibit neural cell development, promote apoptosis, disrupt the homeostasis of the nervous system, and increase the susceptibility to neurodegenerative diseases.

Immunotoxicity
Oxidative stress and apoptosis play key roles in AFB 1 -induced immunotoxicity (Chen et al., 2013). Oral administration of AFB 1 downregulated IFN and TNF in the spleen of mice, increased IL4 levels, and damaged the thymus and spleen, eventually resulting in an impaired immune function (Jebali et al., 2015). AFB 1 exposure in rats increased ROS generation and secretion of pro-inflammatory cytokines (TNFα) in the liver cells (Mohammad et al., 2017), which are conducive HCC development (Qin et al., 2016). AFB 1 exposure affected transcription of key functional genes in human microglia cell line (CHME5) and human monocyte-derived dendritic cells (MDDCs), and increased apoptosis (Mehrzad, Hosseinkhani & Malvandi, 2018a;Mehrzad et al., 2018b). Similarly, AFB 1 treatment decreased the viability of the mouse macrophage RAW264.7 cells in a dose-and time-dependent manner by increasing production of ROS and malondialdehyde (MDA) and decreasing GSH levels. These changes correlated with upregulation of NOS2, TNFα and CXCL2 mRNAs, and downregulation of CD86. AFB 1 -induced oxidative stress in macrophages also impaired the mitochondrial respiratory chain, leading to activation of the inflammatory response pathways (Ma et al., 2021). AFB 1 also decreased the phagocytic capacity of 3D4/21 cells, and induced apoptosis, pro-inflammatory cytokine secretion, DNA damage and oxidative stress. In addition, 3D4/21 cells treated with AFB 1 expressed high levels of DNA methyltransferase DNMT1 and DNMT3a, which led to the activation of the JAK2/STAT3 signaling pathway. Inhibition of p-JAK2 and p-STAT3 by blocking DNMT1 and DNMT3a alleviated AFB 1 -induced immunotoxicity (Zhou et al., 2019). The combination of AFB 1 and ochratoxin A (OTA) increased production of TNFα and IL6 in these cells, and decreased lactate dehydrogenase secretion and the phagocytotic index in a concentration-dependent manner. In addition, the combination treatment significantly decreased the production of GSH, increased ROS levels, and promoted IκBa degradation, NF-κB phosphorylation and nuclear translocation of NF-κB. Thus, AFB 1 and OTA can synergistically aggravate immunotoxicity by activating of the NF-κB signaling pathway (Hou et al., 2018). AFB 1 also impaired the physiological functions of freshly isolated swine alveolar macrophages (SAM), and consumption of AFB 1 -contaminated feed increased the risk of secondary infections in pigs (Pang, Chiang & Chang, 2020). In addition, AFB 1 activated the release of heterophil extracellular traps (HETs) in chicks, and induced the expression of TNFα, IL6 and IL1β, iNOS, COX2, NLRP3, caspase1, caspase3 and caspase11, which resulted in liver and kidney damage (Gao et al., 2022).
Studies show that intake of low doses of AFB 1 in animals adversely affect immune organs, decrease antibody titers and complement activity, and cause lymphoid tissue damage. Monogastric animals, especially poultry and pigs, are more sensitive to AFB 1 -induced immunotoxicity. Broilers exposed to AFB 1 showed increased apoptosis of thymocytes due to mitochondrial and death receptor-mediated signaling pathways, as well as DNA damage . AFB 1 also induced tissue damage, cell cycle arrest (Hu et al., 2018) and apoptosis in the Bursa of Fabricius of broilers, which damaged their immune system (Yuan et al., 2016). Ingestion of AFB 1 -contaminated feed significantly decreased the body weight and lymphocyte activity of pigs, increased the levels of pro-inflammatory cytokines such as TNFα, IL6, IL1β and IFN γ in the spleen, and decreased that of the regulatory factor IL10 (Meissonnier et al., 2008). The immunotoxic effects of AFB 1 in livestock may decrease the efficacy of vaccines and increase disease prevalence. These findings indicate that AFB 1 affects immune cells and organs, and its immunotoxicity depends on the mitochondrial signaling, death receptor, endoplasmic reticulum, apoptosis, cell cycle and inflammatory pathways.

Reproductive toxicity
AFB 1 can impair spermatogenesis through oxidative stress and mitochondria-dependent apoptosis. Mice exposed to AFB 1 show impaired blood-testis barrier due to lower levels of the BTB-related junction protein, increased apoptosis in the testes, and the oxidative stressmediated p38 MAPK signaling pathway, which ultimately affected spermatogenesis (Huang et al., 2021). In addition, AFB 1 exposure decreased spermatogenesis in mice by inducing oxidative stress, decreasing mitochondrial content, and upregulating the pro-apoptotic Bax, p53 and Caspase 3 (Yasin, Mazdak & Mino, 2018). AFB 1 also induced testicular damage and testicular dysfunction in Dorper rams (Lin et al., 2022), and impaired spermatogenesis in white leghorn cockerels (Ashraf et al., 2022). Exposure to low concentrations of AFB 1 for several hours can decrease spermatozoa motility, hyperpolarize mitochondrial membranes, and increase DNA fragmentation (Komsky-Elbaz, Saktsier & Roth, 2018). In addition, AFB 1 interferes with porcine oocyte maturation by inducing epigenetic modifications, oxidative stress, excessive autophagy, and apoptosis (Liu et al., 2015a), and affects early embryo development through oxidative stress, DNA damage, apoptosis, and autophagy (Shin et al., 2018). AFB 1 can cross the placental barrier in humans (Wild et al., 1991), and AFB 1 exposure during pregnancy or lactation can adversely affect the health of the mother as well as the infants. In addition, pregnancy in mice can modulate both phase I and II metabolism, and alter the biological potency of AFB 1 , thereby increasing liver damage (Sriwattanapong et al., 2017). Prenatal exposure to AFB 1 reduces the body weight of neonatal rats, disrupts lipid and hormone levels, and affect the methylation levels of p53 and growth-regulator H19 in the liver and serum. The pathological changes may increase the risk of HCC in the offspring (Rotimi et al., 2021). These studies indicate that exposure of sexually mature animals to AFB 1 affects gamete production, gamete quality, and gamete maturation, and AFB 1 also disrupts embryonic development, posing a long-term health threat to both pregnant animal mothers and offspring. The reproductive toxicity of AFB 1 depends on oxidative stress, DNA damage and repair, apoptosis, autophagy, and epigenetic modification.

MOLECULAR MECHANISM OF AFB 1 TOXICITY AFB 1 and multi-omics analysis
Transcriptomic analyses have helped elucidate the mechanisms underlying the pathological effects of AFB 1 . A total of 1,452 differentially expressed genes (DEGs) have been identified in the liver tissues of AFB 1 -treated versus healthy mice. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were enriched in functions such as cell adhesion, cell proliferation and cell cycle regulation (Xu et al., 2001). In addition, several lncRNAs involved in the regulation of genes related to apoptosis and DNA repair were upregulated following AFB 1 exposure (Shi et al., 2016). Transcriptomic analysis of AFB 1 -exposed rat tissues indicated that the genes affected by AFB 1 were mainly enriched in the p53 signaling pathway, bladder cancerrelated signaling pathways, inflammatory response, antioxidant response, cell proliferation, and DNA repair. Metabolomic analysis showed that AFB 1 dysregulated gluconeogenesis and lipid metabolism (Lu et al., 2013). AFB 1 also induced transcriptomic changes in the genes related to cancer development, apoptosis, inflammation, biological activation, and detoxification in the bovine fetal hepatocyte-derived cell line (BFH12) (Pauletto et al., 2020). Finally, the transcriptomes of AFB 1 -exposed laying hens showed an upregulation of genes involved in hepatic fat deposition and hepatocyte apoptosis, including those related to the mTOR, FoxO, PPAR, fatty acid degradation and fatty acid metabolism pathways (Liu et al., 2020). AFB 1 and the encoding genes AFB 1 can alter the expression levels of oncogenes (such as ras and c-fos) and tumor suppressor genes (such as p53 and survivin) (Su et al., 2004;Duan et al., 2005), and induce genomic instability and mutations by forming DNA adducts, inhibiting DNA repair enzymes and increasing ROS production. It is bio-transformed to AFBO via cytochrome p450 enzymes, which then forms the carcinogenic adducts. In fact, the p53 gene is mutated in the majority of AFB 1 induced HCC cases (Engin & Engin, 2019). In a recent study, transcriptomics and functional genomics identified p53 as the critical transcription factor driving the DNA damage response after exposure to benzo (A) pyrene and AFB 1 (Smit et al., 2017).
The transcription factor Nrf2 regulates antioxidant/stress response genes and detoxification genes, and Nrf2 knockout rats are highly sensitive to AFB 1 (Taguchi et al., 2016). Primary broiler hepatocytes (PBHs) and broiler cardiomyocytes (BCMs) showed a significant decrease in viability, increased mitochondrial dysfunction, ROS generation and high apoptosis rates following AFB 1 treatment, all of which are mediated by the Nrf2 pathway (Liu & Wang, 2016;Wang et al., 2017).
Caveolin-1 (CAV1) is a key mediator of AFB 1 -induced hepatotoxicity. The human hepatocyte L02 cell line showed a marked decline in viability due to increased apoptosis and oxidative stress after AFB 1 exposure, which was accompanied by increased expression of CAV1. The latter mediates AFB 1 -induced oxidative stress through its interaction with Nrf2, leading to the downregulation of cellular antioxidant enzymes and activation of apoptotic pathways. In addition, CAV1 regulates AFB 1 -induced autophagy via the EGFR/PI3K-AKT/mTOR signaling pathway. Taken together, CAV1 plays a crucial role in AFB 1 -induced hepatocyte apoptosis by regulating oxidative stress and autophagy, and is therefore a potential therapeutic target against AFB 1 -related hepatotoxicity (Xu et al., 2020).
AFB 1 induces COX2 expression, promotes mitochondrial autophagy and impairs mitochondrial lipid metabolism in hepatocytes, leading to hepatic steatosis (Ren, Han & Meng, 2020). In addition, AFB 1 can induce apoptosis and trigger an ''eicosanoid and cytokine storm'' in the liver, which can initiate tumor growth. Furthermore, AFB 1generated cellular debris can upregulate COX2, soluble epoxide hydrolase (sEH) and ER stress-response genes (BiP, CHOP and PDI) in macrophages (Fishbein et al., 2020). Meanwhile, the expression of COX2 during pregnancy is crucial, and exposure to AFB 1 may induce physiological changes controlled by COX2 (Zhu, Tan & Leung, 2016).

CONCLUSIONS AND FUTURE DIRECTION
The contaminated of agricultural produce with AFTs is practically unavoidable worldwide. The presence of AFTs in feeds may decrease feed intake, damage health and affect livestock productivity. In addition, the toxic residues in animal products (milk, meat, eggs) may have some adverse effects on human health. Contamination of plant and animal-derived food products by AFB 1 is a major health concern. In this review, we summarized the hepatotoxic, enterotoxic, nephrotoxic, neurotoxic, immune-toxic and gonado-toxic effects of AFB 1 . In addition, the mechanisms underlying the toxicity of AFB 1 , including cell cycle arrest, apoptosis, oxidative stress, ER stress and autophagy, and the effector genes, non-coding RNAs and signaling pathways were also discussed.
The toxicity of AFB 1 is very complex, and is closely related to the dose, exposure duration, administration mode, solvent, species, gender, age, target organs and so on. More comprehensive and systematic tests are needed to ascertain the acute and chronic AFB 1 exposures doses in different species, develop standard dosing methods, and determine Figure 3 The molecular mechanisms of AFB 1 toxicities. Coding genes, non-coding RNAs and signaling pathways involved in AFB 1 -induced toxicity. AFB 1 induces cell cycle arrest, apoptosis, oxidative stress, ER stress and autophagy through multiple genes, non-coding RNAs and signaling pathways. ROS, reactive oxygen species; ER stress, endoplasmic reticulum stress. Made in c BioRender (https://biorender.com/). Full-size DOI: 10.7717/peerj.13850/ fig-3 the quantified dose-response relationship in vitro and in vivo. In addition, the combined effects of AFB 1 with other mycotoxins and with macronutrient deficiency, and well as the underlying mechanisms will be our future research concerns. The relationship between gut microorganisms and sensitivity to AFB 1 , and the impact of intestinal microbiota on AFB 1 metabolism will also be an important research focus. Elucidating the molecular mechanisms underlying the toxicity of AFB 1 can help mitigate or eliminate the toxic effects of AFB 1 by genetic methods, which will also be worth investigating.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the Joint Funds of the National Natural Science Foundation of China (grant numbers: U2004156). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.